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Article

Single Crystal X-Ray Structure for the Disordered Two Independent Molecules of Novel Isoflavone: Synthesis, Hirshfeld Surface Analysis, Inhibition and Docking Studies on IKKβ of 3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-6,7-dimethoxy-4H-chromen-4-one

by
Soon Young Shin
1,
Young Han Lee
1,
Yoongho Lim
2,
Ha Jin Lee
3,
Ji Hye Lee
4,
Miri Yoo
4,
Seunghyun Ahn
4,* and
Dongsoo Koh
4,*
1
Department of Biological Chemistry, Konkuk University, Seoul 05029, Korea
2
Division of Bioscience and Biotechnology, BMIC, Konkuk University, Seoul 05029, Korea
3
Western Seoul Center, Korea Basic Science Institute, Seoul 03759, Korea
4
Department of Applied Chemistry, Dongduk Women’s University, Seoul 02748, Korea
*
Authors to whom correspondence should be addressed.
Crystals 2020, 10(10), 911; https://doi.org/10.3390/cryst10100911
Submission received: 14 September 2020 / Revised: 1 October 2020 / Accepted: 6 October 2020 / Published: 9 October 2020
(This article belongs to the Special Issue Pharmaceutical Crystals (Volume II))
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Versions Notes

Abstract

:
The structure of the isoflavone compound, 3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-6,7-dimethoxy-4H-chromen-4-one (5), was elucidated by 2D-NMR spectra, mass spectrum and single crystal X-ray crystallography. Compound 5, C19H16O6, was crystallized in the monoclinic space group P21/c with the cell parameters; a = 12.0654(5) Å, b =11.0666(5) Å, c = 23.9550(11) Å, β = 101.3757(16)°, V = 3135.7(2) Å3, and Z = 8. The asymmetric unit of compound 5 consists of two independent molecules 5I and 5II. Both molecules exhibit the disorder of each methylene group present in their 1,4-dioxane rings with relative occupancies of 0.599(10) (5I) and 0.812(9) (5II) for the major component A, and 0.401(10) (5I) and 0.188(9) (5II) for the minor component B, respectively. Each independent molecule revealed remarkable discrepancies in bond lengths, bond angles and dihedral angles in the disordered regions of 1,4-dioxane rings. The common feature of the molecules 5I and 5II are a chromone ring and a benzodioxin ring, which are more tilted towards each other in 5I than in 5II. An additional difference between the molecules is seen in the relative disposition of two methoxy substituents. In the crystal, the molecule 5II forms inversion dimers which are linked into chains along an a-axis direction by intermolecular C–H⋯O interactions. Additional C–H⋯O hydrogen bonds connected the molecules 5I and 5II each other to form a three-dimensional network. Hirshfeld surface analysis evaluated the relative intermolecular interactions which contribute to each crystal structure 5I and 5II. Western blot analysis demonstrated that compound 5 inhibited the TNFα-induced phosphorylation of IKKα/β, resulting in attenuating further downstream NF-κB signaling. A molecular docking study predicted the possible binding of compound 5 to the active site of IKKβ. Compound 5 showed an inhibitory effect on the clonogenicity of HCT116 human colon cancer cells. These results suggest that compound 5 can be used as a platform for the development of an anti-cancer agent targeting IKKα/β.

1. Introduction

The NF-κB family of transcription factors plays crucial roles in cellular proliferation, survival, and immune responses. These consist of five members, including c-Rel, p65/RelA, RelB, p50/NF-κB1, and p52/NF-κB2 [1]. The most abundant form is a p65/RelA and p50/NF-κB1. In resting cells, NF-κB dimers remain in an inactive form in the cytoplasm during their association with an inhibitor of κB (IκB) [2]. Upon cellular activation by extracellular stimuli, the upstream IκB kinase (IKK) complex consisting of IKKα, IKKβ, and IKKγ phosphorylates IκB to degrade IκB, allowing the activation of NF-κB [3]. The activated NF-κB complex immediately translocates to the nucleus, thereby regulating the expression of many genes involved in cell survival, cell cycle progression, angiogenesis, invasion, and metastasis [4]. Tumor necrosis factor α (TNFα) is a potent pro-inflammatory cytokine that promotes tumor progression in most types of malignant tumors [5]. It stimulates NF-κB through the activation of IKK [6]. Structure-based drug design (SBDD) and ligand-based drug design (LBDD) are the main streams of computer-aided drug design. Integrated drug design methods have been new trends in this area by combining information from both the ligand and the proteins [7,8]. For the integrated computer-aided studies, three-dimensional molecular structures of proteins and small molecules are prerequisites. Since the three-dimensional x ray structures of the IKKβ protein were revealed [9,10], the development of various inhibitors has been investigated and many inhibitors are commercially available [11,12,13,14,15,16,17,18,19,20,21]. However, it is uncommon for flavonoid compounds to be studied as IKKβ inhibitors [22,23]. Since flavonoids are second metabolites with phytoalexin properties and present in excess amount in plants, many studies and developments have been achieved as dietary supplements [24,25]. Flavonoids have also been reported to exhibit a variety of physiological activities and have been used in the development of a wide range of pharmaceuticals [26,27,28]. Chalcones, flavones, flavonols and isoflavones are diverse forms of flavonoids. In light of our research results, each form of flavonoid has been found to represent unique biological activities [29,30,31,32,33]. In this study, it was intended to investigate the anticancer activity through the IKKβ inhibitory effect after synthesizing the isoflavone compound 5 and revealing its solid-state structure by single crystal x-ray diffraction.

2. Materials and Methods

2.1. General

NMR experiments were carried out on a Bruker Avance 400 spectrometer Q (Bruker, Karlsruhe, Germany). The detailed procedures and parameters for the NMR experiments followed to the methods reported previously [34]. Other general experimental methods were followed by the methods reported previously [35].

2.2. Crystal Structure Determination

Single crystals were obtained by the slow evaporation of the ethanol solution of the isoflavone 5 at ambient temperature. A single crystal of the dimensions 0.393 × 0.305 × 0.134 mm3 was selected and x-ray data were collected at 223 K on a Bruker D8 Venture equipped (Bruker, Madison, EI, USA) with IμS micro-focus sealed tube Mo Kα (λ = 0.71073 Å) and a PHOTON 100 CMOS detector. Bruker SAINT was utilized for the cell refinement and data reduction [36]. The structure was solved by direct methods and refined by full-matrix least-squares on F2 using SHELXTL [37]. Detailed refining methods followed previously reported methods [35], and the outcomes of the crystallographic data collection, structural determination and refinement are summarized in Table 1 (CCDC deposition number 2027508). All relevant information which include bond distances, angles, fractional coordinates, and the equivalent isotropic displacement parameters can be obtained free of charge from the CCDC (Cambridge Crystallographic Data Centre), 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +44 1223 336033; E-mail: [email protected].

2.3. Hirshfeld Surfaces

The Hirshfeld surface analyses were carried out using the program CrystalExplorer 17.5 (University of Western Australia, Perth, Australia) [38,39]. The normalized contact distances (dnorm) were mapped into the Hirshfeld surface, which enabled the visualization of intermolecular interactions by using different colors. In the color scale, the red color denotes closest contact because it indicates the sum of di (the distance from the surface to the nearest nucleus internal to the surface) and de (the distance from the surface to the nearest nucleus external to the surface) and de is shorter than the sum of the relevant van der Waals radii. On the other hand, the white and blue color represent the weak and negligible intermolecular interactions, respectively. The Hirshfeld surfaces and their associated two-dimensional fingerprint plots were used to quantify the various intermolecular interactions in the title compound.

2.4. In Silico Docking with IκB Kinaseβ (IKKβ)

In silico dockings to elucidate the molecular binding mode between isoflavone 5 and IκB kinaseβ (IKKβ) were performed on an Intel Core 2 Quad Q6600 (2.4 GHz) Linux PC with Sybyl 7.3 (Tripos, St. Louis, MO, USA). The three-dimensional structure of IκB kinaseβ (IKKβ) was adopted from a protein data bank deposited as 4KIK.pdb [40]. The detailed experimental procedures followed to the methods previously reported [41].

2.5. Cells and Cell Culture

HCT116 human colon cancer cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD). The cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (HyClone, Logan, UT, USA).

2.6. Western Blot Analysis

HCT116 cells treated with or without 10 ng/mL TNFα (Calbiochem, San Diego, CA, USA) in the presence or absence of compound 5 were lysed in a cell lysis buffer containing 20 mM HEPES (pH 7.2), 1% (v/v) Triton X-100, 10% (v/v) glycerol, 150 mM NaCl, 10 μg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride (PMSF). Protein extracts (20 μg per sample) were separated via 10% (w/v) SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes and incubated with the appropriate primary and secondary antibodies. Primary antibodies against phospho (p)-IKKα/β (Ser176/180), p-IκBα (Ser32), p-p65/RelA (Ser536) were obtained from Cell Signaling Technology (Beverly, MA, USA), and an antibody against glyceraldehyde phosphate dehydrogenase (GAPDH) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All the primary antibodies were diluted to 1:1000 concentration in 50 mM Tris-buffered saline (pH 7.6) containing 5% nonfat dry milk solution and 0.05% Tween-20. The antibody-bound blots were developed using an enhanced chemiluminescence detection system (GE Healthcare, Piscataway, NJ, USA).

2.7. Clonogenic Assay

To measure the long-term growth inhibitory effect of compound 5 against cancer cells, the clonogenic survival assay was performed as reported previously with a minor modification [42]. Briefly, HCT116 human colon cancer cells were plated at a density of 4 × 103 cells per well in 24-well culture plates (BD FalconTM; Becton Dickson Immunocytometry System). After attachment, the cells were incubated in the presence or absence of compound 5 at different concentrations (0, 1, 5, and 10 μM) for 7 days, and fixed with 6% glutaraldehyde, followed by staining with 0.1% crystal violet. Its half-maximal clonogenic growth inhibitory concentration (GI50) was determined using the SigmaPlot software (SYSTAT, Chicago, IL, USA) [43].

3. Results and Discussion

3.1. Synthesis

The title compound 5 was synthesized as shown in Scheme 1 by literature methods [34,44,45]. The final compound 5 was obtained by palladium-catalyzed Suzuki reaction between boronic acid (4) derivative and 3-iodoflavone (3) in three steps from the commercially available starting material.

3.1.1. Synthesis of (E)-3-(dimethylamino)-1-(2-hydroxy-4,5-dimethoxyphenyl)prop-2-en-1-one (2)

The previously reported literature procedures were used, but starting with 2-hydroxy-4,5-dimethoxyacetophenone (1) [34,44]. 1H NMR (400 MHz, DMSO) δ 14.76 (s, 1H), 7.83 (d, J = 12.0 Hz, 1H), 7.31 (s, 1H), 6.41 (s, 1H), 5.85 (d, J = 12.0 Hz, 1H), 3.78 (s, 3H), 3.75 (s, 3H), 3.17 (s, 3H), 2.99 (s, 3H). 13C NMR (100 MHz, DMSO) δ 189.35, 159.65, 154.95, 154.74, 141.13, 111.98, 111.41, 100.78, 89.38, 56.95, 55.71, 44.96, 37.54.

3.1.2. Synthesis of 3-iodo-6,7-dimethoxy-4H-chromen-4-one (3)

The slightly modified literature procedures were used, but starting with previously obtained enamine 2 [34,44]. After the completion of the reaction, the precipitate was formed. The resulting solid was filtered and was washed with cold methanol. The solid compound of iodoflavone (3) was pure and used for the next reaction without further purifications. 1H NMR (400 MHz, DMSO) δ 8.73 (s, 1H), 7.37 (s, 1H), 7.31 (s, 1H), 3.90 (s, 3H), 3.85 (s, 3H). 13C NMR (101 MHz, DMSO) δ 171.82, 158.31, 154.69, 152.04, 147.91, 114.46, 104.22, 100.44, 86.58, 56.62, 56.00.

3.1.3. Synthesis of 3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-6,7-dimethoxy-4H-chromen-4-one (5)

The previously reported literature procedures were used but starting with the 3-iodo flavone compound (3) [34,45]. For the complete assignment for the proton atoms and carbon atoms, additional two-dimensional NMR such as HMBC (heteronuclear multiple bond correlation), HMQC (heteronuclear multiple-quantum correlation) and TOCSY (total correlation spectroscopy) were performed (Supplementary Materials). 1H NMR (400 MHz, DMSO-d6) δ 8.34 (s, 1H, H-2), 7.47 (s, 1H, H-5), 7.16 (s, 1H, H-8), 7.15 (d, 1H, H-2′, J = 2.1 Hz), 7.06 (dd, 1H, H-6′, J = 8.4, 2.1 Hz), 6.88 (d, 1H, H-5′, J = 8.4 Hz), 4.27 (s, 4H, 3′, 4′-CH2), 3.93 (s, 3H, 7-OCH3), 3.88 (s, 3H, 6-OCH3); 13C NMR (400 MHz, DMSO-d6) δ 173.9 (C-4), 154.2 (C-7), 152.8 (C-2), 151.5 (C-9), 147.4 (C-6), 143.0 (C-4′), 142.8 (C-3′), 124.9 (C-1′), 122.4 (C-3), 121.5 (C-6′), 117.3 (C-2′), 117.0 (C-10), 116.4 (C-5′), 104.4 (C-5), 100.1 (C-8), 63.90 (C-3′), 63.86 (C-4′), 56.1 (7-OCH3), 55.7 (6-OCH3). HR/MS (m/z): Calcd. for (M+H)+: 341.0980; Found: 341.1032.

3.2. Crystal Structure of Isoflavone Compound 5

The asymmetric unit of compound 5 consists of two independent molecules 5I (C1–C19) and 5II (C20–C38). In each independent molecules, two methylene groups C18–C19 (5I) and C37–C38 (5II) in corresponding 1, 4-dioxane rings are disordered over two positions with relative occupancies of 0.599(10) (5I) and 0.812(9) (5II) for the major component A, and 0.401(10) (5I) and 0.188(9) (5II) for the minor component B, respectively (Figure 1A). From a macroscopic point of view, two independent molecules 5I and 5II are roughly superimposed over each other as shown in Figure 1B.
There are relative differences in the corresponding bond distances, bond angles, and torsional angles among the conformers 5IA–5IIB. In the C1–C19 molecule (5I), the two dioxane rings at major component 5IA and minor component 5IB are both in the half-chair conformations. The atom C18A shows maximum deviation from the dioxane ring of C14–C15–O6–C19A–C18A–O5 (r.m.s. deviation = 0.212 Å) by 0.351 Å in the major component 5IA. The atom C19B shows maximum deviation from the ring of C14–C15–O6–C19B–C18B–O5 (rmsd = 0.201 Å) by 0.338 Å in the minor component 5IB. The dihedral angle formed between plane of the dimethoxy-substituted benzene ring (C2–C7; rmsd = 0.004 Å) and a plane of dioxane-attached benzene ring (C12–C17; rmsd = 0.003 Å) is 47.45(4)°. The methoxy groups are slightly twisted from the benzene ring by torsion angle of C3–C4–O3–C10 = 10.6(2)° at C4 and C6–C5–O4–C11 = −5.5(2)o at C5, respectively, in the molecule 5I (Figure 2).
Both the dioxane ring (C33–C34–O11–C38A–C37A–O12 (rmsd 0.204 Å)) of the major component and the dioxane ring (C33–C34–O11–C38B–C37B–O12 (rmsd 0.226 Å)) of a minor component lie in the half-chair conformations in molecule 5II as well. The maximum deviations from each dioxane ring are 0.332 Å at C37A, and 0.383 Å at C38B, respectively. For the independent molecule 5II (C20–C38), the dihedral angle formed between the corresponding two benzene rings of (C21–C26; rmsd = 0.004 Å) and (C31–C36; rmsd = 0.003Å) is 34.82(2)°, which is slightly less twisted compared to molecule 5I. In addition, the methoxy groups are almost coplanar with the benzene ring by the torsion angle of C25–C24–O10–C30 = −1.5(3)° at C23 and C22–C23–O9–C29 = 2.6(2)o at C24, respectively (Figure 3).
There are distinctive discrepancies in the bond lengths and bond angles in the distorted regions of molecules 5IA, 5IB, 5IIA and 5IIB. Comparing the bond lengths between two conformers (A and B) of each independent molecule (5I, 5II), the molecule 5I revealed significant difference in bond lengths around the disordered area. For the major conformer 5IA, the bond lengths were O(5)–C(18A) = 1.412(4) Å, C(18A)–C(19A) = 1.506(8) Å, C(19A)–O(6) =1.495(4) Å, respectively, and for the minor conformers 5IB, those are O(5)–C(18B) = 1.513(7) Å, C(18B)–C(19B) = 1.458(13) Å, C(19B)–O(6) =1.402(6) Å, respectively. When they are compared in an inter-molecular manner, the bond lengths of O(5)–C(18B) and C(18B)–C(19B) in crystal 5I are longer than those of the corresponding O(11)–C(37B) and C(37B)–C(38B) in crystal 5II. Bond angles C(38B)–C(37B)–O(11) = 110.9(15)° and C(37B)–C(38B)–O(12) = 105.2(16)° in molecule 5IIB are smaller than the corresponding bond angles O(5)–C(18B)–C(19B) = 113.2(7)° and O(6)–C(19B)–C(18B) = 106.5(8)° in molecule 5IB (Table 2).
In the crystal, the pairs of the intermolecular C37–H37B···O11 hydrogen bonds form inversion dimers with R22(6) graph-set motifs. The dimers are linked into chains along the a axis direction by pairs of the C38–H38B···O9 hydrogen bonds in the molecule II (Figure 4, Table 3).
The two molecules 5I and 5II are connected to each other by intermolecular hydrogen bonds C11–H11B···O12 and C29–H29B···O1 to form an ac-plane from two-dimensional supramolecules (Figure 5, Table 3).

3.3. Hirshfeld Surface analysis of Compound 5

In order to quantify the intermolecular interactions in the crystals of the titled compound 5, a Hirshfeld surface (HS) analysis was carried out. Based on the Hirshfeld analysis on all conformers, two independent molecules (5I and 5II) showed different dnorm, shape index (SI) and curvedness, however, each set of conformers A and B revealed the same Hirshfeld analysis results [46]. The 3D Hirshfeld surfaces of two independent molecules (5I and 5II) were illustrated in Figure 6A,B, which maps dnorm, shape index and curvedness. The deep red spots on the dnorm Hirshfeld surfaces of each molecule represent the close contact interactions, which are mainly responsible for the significant intermolecular C–H···O interactions. Shape index and curvedness can also be used to identify the characteristic packing modes. The shape indexes of 5I and 5II show red concave regions on the surface around the acceptor atoms and blue regions around the donor H atoms. The maps of curvedness for 5I and 5II show no flat surface patches representing that there are no stacking interactions between the molecules [47].
According to two-dimensional fingerprint plots analysis, the dominant interaction in each molecule 5I and 5II originates from H···H contacts, which are the major contributors of 43.5% and 42.5% to the total Hirshfeld surface, respectively. The contribution from the O···H/H···O contacts of 25.1% and 29.1% of each molecule 5I and 5II is represented by a pair of sharp spikes that are characteristic of hydrogen-bonding interactions. Other meaningful interactions include C···H/H···C with contributions of 17.8% and 16.7% from 5I and 5II, respectively (Figure 7A–H).
The overall contribution to the total Hirshfeld surface is illustrated in Figure 8.

3.4. In Silico Docking with IKKβ

The docking calculations were carried out using the protein structure of IKKβ (The Protein Data Bank code; 4KIK.pdb). Using the Sybyl program, the apo-protein of IKKβ was obtained by removing the original ligand K252a contained in 4KIK.pdb. The original ligand K252a was again docked to the apo-protein, to confirm that the flexible docking procedure worked well. Through a flexible docking procedure repeated 30 times, 30 complexes between apo-protein and K252a were obtained. Their binding energy ranged from −28.44 to −10.24 kcal/mol and their binding poses were good to be comparable with 4KIK.pdb.The binding pocket of IKKβ was determined using the LigPlot software as previously reported [48]. They are composed of 16 residues; 14 residues, namely Leu21, Gly22, Thr23, Val25, Ala42, Lys44, Glu61, Val74, Met96, Tyr98, Glu149, Asn150, Ile165, and Asp166 are involved in hydrophobic interactions, and two residues, Glu97 and Cys99 are involved in hydrogen bonds. Using the three-dimensional structure of compound 5 obtained in this study, docking with apo-protein was performed in the same way as the original ligand. The binding energy generated by the 30 iterations ranged from −15.92 to −13.09 kcal/mol. The interactions between IKKβ and compound 5 were analyzed using the LigPlot progam. Six residues including Thr23, Val29, Glu61, Met65, Met96, and Ile165 showed the hydrophobic interactions with the ligand and three residues including Gly27, Lys44, and Asp166 formed hydrogen bonds (H bonds) with the ligand (Figure 9A). The binding pocket of compound 5 resided in IKKβ was visualized using the PyMOL program (PyMOL Molecular Graphics System, version 1.0r1, Schrödinger, LLC, Portland, OR, USA). Isoflavone compound 5 in IKKβ exhibited a slightly different binding pattern from those of the original ligand K252a. However, both isoflavone 5 and original ligand K252a have been shown to bind well at the active site of the IKKβ protein (Figure 9B).

3.5. Effect of Compound 5 on the Inhibition of IKK Pathway

To validate the in silico docking prediction that IKKβ is a target for compound 5, we tested the effect of compound 5 on the inhibition of the IKK signaling pathway using a cell-based kinase assay. We confirmed that the phosphorylation of IKKα/β and its downstream targets IκB and p65/RelA was rapidly induced within 10 min and then gradually decreased upon 10 ng/mL TNFα stimulation (Figure 10A). Under this experimental condition, we determined whether compound 5 inhibited the TNFα-induced IKK activity. HCT116 cells were pre-treated with different concentrations of compound 5 (0, 50, 100 μM) for 30 min and then stimulated with 10 ng/mL TNFα for 10 min. We observed that pre-treatment with compound 5 dose-dependently reduced phosphorylation of IKKα/β and its downstream target IκB and p65/RelA, induced by TNFα (Figure 10B). These data suggest that compound 5 inhibits TNFα-induced NF-κB activation through the targeting of IKK.

3.6. Effect of Compound 5 on the Inhibition of Clonogenicity of HCT116 Cells

To support the idea that NF-κB inhibition through IKK targeting by compound 5 exerts an antiproliferation effect, we tested the inhibitory activity of compound 5 on the clonogenicity of HCT116 human colon cancer cells. Clonogenic assay is an in vitro non-destructive method known to reflect the in vivo evaluation of anticancer drugs. Treatment with compound 5 for 7 days resulted in a dose-dependent loss of clonogenicity of HCT116 cells (Figure 11). Its GI50 value was determined to be 17.2 μM. These data suggest that IKK targeting by compound 5 reduced the individual cell ability to proliferate into viable colonies.
NF-κB is constitutively activated in most cancer cells. The inhibition of NF-κB in cancer cells causes the induction of the cell cycle arrest and apoptosis. Therefore, pharmacological NF-κB inhibitors have been widely used for cancer prevention and therapy. The full activation of p65/RelA NF-κB is necessary for the release of the NF-κB complex from IκB. IKK phosphorylates IκB on Serine-32, triggering the proteasome-dependent proteolysis of IκB and releasing NF-κB from IκB. IKK also phosphorylates and degrades the Forkhead transcription factor FOXO3a that is involved in cell cycle arrest and apoptosis [49] and activates insulin receptor substrate (IRS) to impair insulin signaling [50], suggesting that IKK inhibition can exhibit multiple anticancer activities in addition to inhibiting NF-κB. Previous study has demonstrated that the most effective and selective approach for NF-κB inhibition might be offered by the IKK inhibitor [51], suggesting that the selective targeting of IKK is a promising therapeutic strategy for anticancer drug development. In this study, we identified compound 5 that inhibits the NF-κB signaling pathway through targeting the upstream kinase IKKβ.

4. Conclusions

The title compound 5, C19H16O6, was crystallized in the monoclinic space group P21/c and consists of two independent molecules 5I and 5II. Both molecules exhibit the disorder of each methylene groups present in the 1,4-dioxane ring with occupancies 0.6289 (17) and 0.3711 (17), respectively. Based on the Hirshfeld analysis, two independent crystals (5I and 5II) showed differences in the dnorm, shape index (SI), curvedness and the overall contribution to the total Hirshfeld surface. In the crystal, the molecule 5II forms inversion dimers which are linked into chains along the a axis direction by intermolecular C–H···O interactions. Western blot analysis demonstrated that compound 5 inhibited the TNFα-induced phosphorylation of IKKα/β. A molecular docking study predicted the possible binding of compound 5 to the active site of IKKβ. We found that isoflavone derivative 5 inhibited the TNFα-induced phosphorylation of IKKα/β and its downstream IκB and p65/RelA NF-κB, which suggest that compound 5 can be used as a platform for the development of anti-cancer agent targeting IKKα/β.

Supplementary Materials

The following are available online at https://www.mdpi.com/2073-4352/10/10/911/s1, CIF file and NMR spectra for reaction intermediates compounds 2, 3, and final product compound 5: Figure S1: 1H NMR spectrum of compound 2; Figure S2: 13C-NMR spectrum of compound 2; Figure S3: 1H-NMR spectrum of compound 3.; Figure S4: 13C-NMR spectrum of compound 3; Figure S5: 1H-NMR spectrum of compound 5; Figure S6: 13C-NMR spectrum of compound 5; Figure S7: 2D-HMBC spectrum of compound 5; Figure S8: 2D-HMQC spectrum of compound 5; Figure S9: 2D-TOSCY spectrum of compound 5.

Author Contributions

Conceptualization, D.K. and S.Y.S.; investigation, S.A., J.H.L. and M.Y.; validation, Y.H.L., H.J.L. and Y.L.; writing—original draft, S.A., D.K. writing—review and editing, S.Y.S. All authors have read and agreed to the published version of the manuscript.

Funding

The authors acknowledge financial support from the Basic Science Research Program (award No. NRF- 2019R1F1A1058747). S.Y. Shin was supported by the KU Research Professor Program of Konkuk University.

Conflicts of Interest

The authors declare no conflict of interest.

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Crystals 10 00911 sch001 550
Scheme 1. Synthetic procedures for the title compound 5.
Scheme 1. Synthetic procedures for the title compound 5.
Crystals 10 00911 sch001
Crystals 10 00911 g001 550
Figure 1. (A) Molecular structure of the title compound 5 with atomic labelling. The asymmetric part of the unit cell incorporates two independent molecules 5I (C1–C19) and 5II (C20–C38). Displacement ellipsoids are drawn at the 30% probability level. The minor component of the disordered moiety is drawn with open bonds. (B) An overlay diagram of two independent crystals 5I (orange color) and 5II (blue color) of title compound 5. H atoms are omitted for clarity.
Figure 1. (A) Molecular structure of the title compound 5 with atomic labelling. The asymmetric part of the unit cell incorporates two independent molecules 5I (C1–C19) and 5II (C20–C38). Displacement ellipsoids are drawn at the 30% probability level. The minor component of the disordered moiety is drawn with open bonds. (B) An overlay diagram of two independent crystals 5I (orange color) and 5II (blue color) of title compound 5. H atoms are omitted for clarity.
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Crystals 10 00911 g002 550
Figure 2. View of the molecular structure of the independent molecule 5I with the atom label including all hydrogens. It shows a disordered structure in the 1, 4-dioxane rings. Displacement ellipsoids are drawn at the 30% probability level.
Figure 2. View of the molecular structure of the independent molecule 5I with the atom label including all hydrogens. It shows a disordered structure in the 1, 4-dioxane rings. Displacement ellipsoids are drawn at the 30% probability level.
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Crystals 10 00911 g003 550
Figure 3. View of the molecular structure of independent molecule 5II with the atom label including all hydrogens. It shows disordered structure in 1, 4-dioxane rings. Displacement ellipsoids are drawn at the 30% probability level.
Figure 3. View of the molecular structure of independent molecule 5II with the atom label including all hydrogens. It shows disordered structure in 1, 4-dioxane rings. Displacement ellipsoids are drawn at the 30% probability level.
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Crystals 10 00911 g004 550
Figure 4. Pairs of hydrogen bonds form an inversion dimer (orange dashed line) which are linked into chains along the a-axis.
Figure 4. Pairs of hydrogen bonds form an inversion dimer (orange dashed line) which are linked into chains along the a-axis.
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Crystals 10 00911 g005 550
Figure 5. A view along the b axis of the crystal packing of compound 5. The molecule I and II are linked via intermolecular hydrogen bonds C11–H11B···O12 and C29–H29B···O1. For clarity, the hydrogen atoms not involved in H bonds are omitted.
Figure 5. A view along the b axis of the crystal packing of compound 5. The molecule I and II are linked via intermolecular hydrogen bonds C11–H11B···O12 and C29–H29B···O1. For clarity, the hydrogen atoms not involved in H bonds are omitted.
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Crystals 10 00911 g006 550
Figure 6. (A) Hirshfeld surfaces of molecule 5I mapped with dnorm, shape index and curvedness. (B) Hirshfeld surfaces of molecule 5II mapped with dnorm, shape index and curvedness.
Figure 6. (A) Hirshfeld surfaces of molecule 5I mapped with dnorm, shape index and curvedness. (B) Hirshfeld surfaces of molecule 5II mapped with dnorm, shape index and curvedness.
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Crystals 10 00911 g007 550
Figure 7. Two-dimensional fingerprint plots of the most important intermolecular contacts in each molecule I and II. For I: full (A) and resolved into O···H (B), H···H (C), C···H (D). For II: full (E) and resolved into O···H (F), H···H (G), C···H (H).
Figure 7. Two-dimensional fingerprint plots of the most important intermolecular contacts in each molecule I and II. For I: full (A) and resolved into O···H (B), H···H (C), C···H (D). For II: full (E) and resolved into O···H (F), H···H (G), C···H (H).
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Crystals 10 00911 g008 550
Figure 8. Summary of the overall detailed intermolecular interactions and their contribution to each crystal structure I and II. For I; H···H; 43.5%, O···H; 25.1%, O···C; 6.4%, C···H; 17.8%, C···C; 5.7%, O···O; 1.5%, For II; H···H; 42.5%, O···H; 29.1%, O···C; 5.6%, C···H; 16.3%, C···C; 5.7%, O···O; 0.8%.
Figure 8. Summary of the overall detailed intermolecular interactions and their contribution to each crystal structure I and II. For I; H···H; 43.5%, O···H; 25.1%, O···C; 6.4%, C···H; 17.8%, C···C; 5.7%, O···O; 1.5%, For II; H···H; 42.5%, O···H; 29.1%, O···C; 5.6%, C···H; 16.3%, C···C; 5.7%, O···O; 0.8%.
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Crystals 10 00911 g009 550
Figure 9. (A) The residues participating in the binding sites of the compound 5-IKKβ complex analyzed by using the LigPlot program. (B) Three-dimensional image of the IKKβ and compound 5 complex, where compound 5 is colored in green and the original ligand K252a contained in IKKβ is colored in red.
Figure 9. (A) The residues participating in the binding sites of the compound 5-IKKβ complex analyzed by using the LigPlot program. (B) Three-dimensional image of the IKKβ and compound 5 complex, where compound 5 is colored in green and the original ligand K252a contained in IKKβ is colored in red.
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Crystals 10 00911 g010 550
Figure 10. Effect of compound 5 on the inhibition of the IKK signaling pathway in HCT116 colon cancer cells. (A) HCT116 cells were starved with 0.5% fetal bovine serum for 24 h, followed by stimulation with 10 ng/mL TNFα for the indicated times. Whole-cell lysates were prepared, and Western blotting was performed using the phospho-specific antibody against IKKα/β (Ser176/180), IκBα (Ser32), and p65/RelA NF-κB (Ser536). Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as an internal control. (B) HCT116 cells were starved with 0.5% FBS for 24 h, followed by pre-treatment with compound 5 (5 or 20 μM) 30 min before stimulation with 10 ng/mL TNFα. After 10 min, whole-cell lysates were prepared, and Western blotting was performed using the phospho-specific antibody against IKKα/β (Ser176/180), IκBα (Ser32), and p65/RelA NF-κB (Ser536). GAPDH was used as an internal control.
Figure 10. Effect of compound 5 on the inhibition of the IKK signaling pathway in HCT116 colon cancer cells. (A) HCT116 cells were starved with 0.5% fetal bovine serum for 24 h, followed by stimulation with 10 ng/mL TNFα for the indicated times. Whole-cell lysates were prepared, and Western blotting was performed using the phospho-specific antibody against IKKα/β (Ser176/180), IκBα (Ser32), and p65/RelA NF-κB (Ser536). Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as an internal control. (B) HCT116 cells were starved with 0.5% FBS for 24 h, followed by pre-treatment with compound 5 (5 or 20 μM) 30 min before stimulation with 10 ng/mL TNFα. After 10 min, whole-cell lysates were prepared, and Western blotting was performed using the phospho-specific antibody against IKKα/β (Ser176/180), IκBα (Ser32), and p65/RelA NF-κB (Ser536). GAPDH was used as an internal control.
Crystals 10 00911 g010
Crystals 10 00911 g011 550
Figure 11. Effect of compound 5 on the inhibition of clonogenicity of HCT116 colon cancer cells.
Figure 11. Effect of compound 5 on the inhibition of clonogenicity of HCT116 colon cancer cells.
Crystals 10 00911 g011
Table
Table 1. Crystal data and structure refinement for compound 5.
Table 1. Crystal data and structure refinement for compound 5.
CCDC deposit number2027508
Empirical formulaC19 H16 O6
Formula weight340.32
Temperature223(2) K
Wavelength0.71073 Å
Crystal systemMonoclinic
Space groupP21/c
Unit cell dimensionsa = 12.0654(5) Å
b = 11.0666(5) Å
c = 123.9550(11) Å
β = 101.3757(16)°.
Volume3135.7(2) Å3
Z8
Density (calculated)1.442 Mg/m3
Absorption coefficient0.108 mm−1
F(000)1424
Crystal size0.393 × 0.305 × 0.134 mm3
Theta range for data collection1.722 to 28.347°
Index ranges−16 ≤ h ≤ 16, −14 ≤ k ≤ 14, −31 ≤ l ≤ 31
Reflections collected102751
Independent reflections7798 [R(int) = 0.0604]
Completeness to theta = 25.242°99.7 %
Max. and min. transmission0.7457 and 0.6970
Refinement methodFull-matrix least-squares on F2
Data/restraints/parameters7798/12/492
Goodness-of-fit on F21.063
Final R indices [I>2sigma(I)]R1 = 0.0430, wR2 = 0.1004
R indices (all data)R1 = 0.0666, wR2 = 0.1179
Largest diff. peak and hole0.226 and −0.206 e.Å−3
Table
Table 2. Selected bond lengths (Å) and bond angles (°) in the distorted regions of crystal 5IA, 5IB, 5IIA and 5IIB, which show distinctive discrepancy. Torsional angles (°) were shown for the difference in the methoxy group substitutions.
Table 2. Selected bond lengths (Å) and bond angles (°) in the distorted regions of crystal 5IA, 5IB, 5IIA and 5IIB, which show distinctive discrepancy. Torsional angles (°) were shown for the difference in the methoxy group substitutions.
III
O(5)–C(18A)1.412(4)O(11)–C(37A)1.447(3)
O(5)–C(18B)1.513(7)O(11)–C(37B)1.422(14)
C(18A)–C(19A)1.506(8)C(37A)–C(38A)1.507(6)
C(18B)–C(19B)1.458(13)C(37B)–C(38B)1.45(3)
C(19A)–O(6)1.495(4)C(38A)–O(12)1.452(3)
C(19B)–O(6)1.402(6)C(38B)–O(12)1.449(15)
O(5)–C(18A)–C(19A)107.5(4)O(11)–C(37A)–C(38A)109.2(3)
C(18A)–C(19A)–O(6)109.5(4)O(12)–C(38A)–C(37A)108.8(3)
O(5)–C(18B)–C(19B)113.2(7)C(38B)–C(37B)–O(11)110.9(15)
O(6)–C(19B)–C(18B)106.5(8)C(37B)–C(38B)–O(12)105.2(16)
C(15)–O(6)–C(19A)111.59(18)C(34)–O(12)–C(38A)113.50(15)
C(15)–O(6)–C(19B)113.9(3)C(34)–O(12)–C(38B)108.9(5)
C(3)–C(4)–O(3)–C(10)10.6(3)C(22)–C(23)–O(9)–C(29)2.6(2)
C(6)–C(5)–O(4)–C(11)−5.5(2)C(25)–C(24)–O(10)–C(30)−1.5(3)
Table
Table 3. Intermolecular hydrogen bonds involved in the crystal packing of compound 5 (Å and °).
Table 3. Intermolecular hydrogen bonds involved in the crystal packing of compound 5 (Å and °).
D–H…Ad(D–H)d(H…A)d(D…A)<(DHA)
C(29)–H(29B)…O(1)#10.972.433.396(2)172.2
C(38A)–H(38B)…O(9)#20.982.463.396(3)157.8
C(10)–H(10B)…O(7)#30.972.483.424(2)165.6
C(37A)–H(37B)…O(11)#40.982.593.071(4)110.7
Symmetry transformations used to generate equivalent atoms: #1 x+1, y, z; #2 x−1, -y+3/2, z−1/2; #3 x, −y+3/2, z+1/2; #4 −x, −y+1, −z.

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Shin, S.Y.; Lee, Y.H.; Lim, Y.; Lee, H.J.; Lee, J.H.; Yoo, M.; Ahn, S.; Koh, D. Single Crystal X-Ray Structure for the Disordered Two Independent Molecules of Novel Isoflavone: Synthesis, Hirshfeld Surface Analysis, Inhibition and Docking Studies on IKKβ of 3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-6,7-dimethoxy-4H-chromen-4-one. Crystals 2020, 10, 911. https://doi.org/10.3390/cryst10100911

AMA Style

Shin SY, Lee YH, Lim Y, Lee HJ, Lee JH, Yoo M, Ahn S, Koh D. Single Crystal X-Ray Structure for the Disordered Two Independent Molecules of Novel Isoflavone: Synthesis, Hirshfeld Surface Analysis, Inhibition and Docking Studies on IKKβ of 3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-6,7-dimethoxy-4H-chromen-4-one. Crystals. 2020; 10(10):911. https://doi.org/10.3390/cryst10100911

Chicago/Turabian Style

Shin, Soon Young, Young Han Lee, Yoongho Lim, Ha Jin Lee, Ji Hye Lee, Miri Yoo, Seunghyun Ahn, and Dongsoo Koh. 2020. "Single Crystal X-Ray Structure for the Disordered Two Independent Molecules of Novel Isoflavone: Synthesis, Hirshfeld Surface Analysis, Inhibition and Docking Studies on IKKβ of 3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-6,7-dimethoxy-4H-chromen-4-one" Crystals 10, no. 10: 911. https://doi.org/10.3390/cryst10100911

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